Aberrant Activation of Notch1 Signaling in Glomerular Endothelium Induces Albuminuria.

RATIONALE: Glomerular capillaries are lined with a highly specialized fenestrated endothelium and contribute to the glomerular filtration barrier. The Notch signaling pathway is involved in regulation of glomerular filtration barrier, but its role in glomerular endothelium has not been investigated due to the embryonic lethality of animal models with genetic modification of Notch pathway components in the endothelium. OBJECTIVE: To determine the effects of aberrant activation of the Notch signaling in glomerular endothelium and the underlying molecular mechanisms. METHODS AND RESULTS: We established the ZEG-NICD1 (notch1 intracellular domain)/Tie2-tTA/Tet-O-Cre transgenic mouse model to constitutively activate Notch1 signaling in endothelial cells of adult mice. The triple transgenic mice developed severe albuminuria with significantly decreased VE-cadherin (vascular endothelial cadherin) expression in the glomerular endothelium. In vitro studies showed that either NICD1 (Notch1 intracellular domain) lentiviral infection or treatment with Notch ligand DLL4 (delta-like ligand 4) markedly reduced VE-cadherin expression and increased monolayer permeability of human renal glomerular endothelial cells. In addition, Notch1 activation or gene knockdown of VE-cadherin reduced the glomerular endothelial glycocalyx. Further investigation demonstrated that activated Notch1 suppression of VE-cadherin was through the transcription factors SNAI1 (snail family transcriptional repressor 1) and ERG (Ets related gene), which bind to the -373 E-box and the -134/-118 ETS (E26 transformation-specific) element of the VE-cadherin promoter, respectively. CONCLUSIONS: Our results reveal novel regulatory mechanisms whereby endothelial Notch1 signaling dictates the level of VE-cadherin through the transcription factors SNAI1 and ERG, leading to dysfunction of glomerular filtration barrier and induction of albuminuria. Graphic Abstract: A graphic abstract is available for this article.

Glomerular Endothelial Cells: Assessment of Barrier Properties In Vitro.

Endothelial cells form the inner lining of all blood vessels and play a vital role in regulating vascular permeability. This applies to the circulation in general and also to specific capillary beds including the renal glomerular capillaries. Endothelial dysfunction, including increased permeability, is a key component of diabetes-induced organ damage. Endothelial cells together with their glycocalyx, grown on porous membranes, provide an excellent model to study endothelial permeability properties. Here we describe the measurement of two characteristics of glomerular endothelial cell (GEnC) monolayers: electrical resistance and macromolecular passage. Trans-endothelial electrical resistance provides a measure of small-pore pathways across the endothelium and provides an index of monolayer confluence and cell-cell junction integrity. Measurement of macromolecular passage provides an index of large-pore pathways and use of labeled albumin provides direct relevance to the clinically important parameter of albuminuria. The combination of the two approaches provides a fantastic tool to elucidate endothelial barrier function in vitro including in response to cytokines, pathological stimuli, and potential therapeutic agents.

Proteomic analysis of the kidney filtration barrier--Problems and perspectives.

Diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. The kidney filter is localized within the renal glomeruli, small microvascular units that are responsible for ultrafiltration of about 180 liters of primary urine every day. The renal filter consists of three layers, fenestrated endothelial cells, glomerular basement membrane, and the podocytes, terminally differentiated, arborized epithelial cells. This review demonstrates the use of proteomics to generate insights into the regulation of the renal filtration barrier at a molecular level. The advantages and disadvantages of different glomerular purification methods are examined, and the technical limitations that have been significantly improved by in silico or biochemical approaches are presented. We also comment on phosphoproteomic studies that have generated considerable molecular-level understanding of the physiological regulation of the kidney filter. Lastly, we conclude with an analysis of urinary exosomes as a potential filter-derived resource for the noninvasive discovery of glomerular disease mechanisms.

MAGI-2 is critical for the formation and maintenance of the glomerular filtration barrier in mouse kidney.

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a tight junction protein in epithelial tissues. We previously reported the detailed expression patterns of MAGI-2 in mouse tissues, including kidney podocytes, based on results obtained from Venus knock-in mice for Magi2 locus. In the present study, homozygous deletion of the Magi2 gene in mice caused neonatal lethality, which was explained by podocyte morphological abnormalities and anuria. Immunohistological analysis showed that loss of MAGI-2 function induced a significant decrease in nephrin and dendrin at the slit diaphragm of the kidney, although other components of the slit diaphragm were unchanged. Furthermore, nuclear translocation of dendrin was observed in the podocytes of the MAGI-2-null mutants, along with enhanced expression of cathepsin L, which is reported to be critical for rearrangement of the actin cytoskeleton in podocytes. Expression analysis of the null mutants showed that loss of MAGI-2 function induces abnormal expression of various types of adhesion-related molecules. The present study is the first to demonstrate that MAGI-2 has a critical role in maintaining the functional structure of the slit diaphragm and that this molecule has an essential role in the functioning of the kidney filtration barrier.

Podocyte-specific GLUT4-deficient mice have fewer and larger podocytes and are protected from diabetic nephropathy.

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.

Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies.

Drosophila nephrocytes are functionally homologous to vertebrate kidney podocytes. Both share the presence of slit diaphragms that function as molecular filters during the process of blood and haemolymph ultrafiltration. The protein components of the slit diaphragm are likewise conserved between flies and humans, but the mechanisms that regulate slit diaphragm dynamics in response to injury or nutritional changes are still poorly characterised. Here, we show that Dumbfounded/Neph1, a key diaphragm constituent, is a target of the Src kinase Src64B. Loss of Src64B activity leads to a reduction in the number of diaphragms, and this effect is in part mediated by loss of Dumbfounded/Neph1 tyrosine phosphorylation. The phosphorylation of Duf by Src64B, in turn, regulates Duf association with the actin regulator Dock. We also find that diaphragm damage induced by administration of the drug puromycin aminonucleoside (PAN model) directly associates with Src64B hyperactivation, suggesting that diaphragm stability is controlled by Src-dependent phosphorylation of diaphragm components. Our findings indicate that the balance between diaphragm damage and repair is controlled by Src-dependent phosphorylation of diaphragm components, and point to Src family kinases as novel targets for the development of pharmacological therapies for the treatment of kidney diseases that affect the function of the glomerular filtration barrier.

Hydrogen peroxide induces dimerization of protein kinase G type Iα subunits and increases albumin permeability in cultured rat podocytes.

Podocytes help regulate filtration barrier permeability in the kidneys. They express contractile proteins that are characteristic of smooth muscle cells as well as receptors for vasoactive factors such as angiotensin II and atrial natriuretic peptide (ANP). The later one generates intracellular cGMP, with subsequent activation of cGMP-dependent protein kinase; PKG (isoform PKGIα and PKGIß). In this study, we asked whether hydrogen peroxide (H(2)O(2)), a physiological vasorelaxing factor, affected podocyte permeability and the podoctye PKGIα signaling pathway. Expression of PKGIα was confirmed in cultured rat podocytes using RT-PCR, immunofluorescence, and Western blotting. Exposure of podocytes to exogenous H(2)O(2) (100 µM) in non-reducing conditions increased the formation of PKGIα interprotein disulfide bonds, affected the phosphorylation of PKG target proteins, namely MYPT1 (maximal increase of about 57% at 30 min) and MLC (maximal decrease of about 62% at 10 min). Furthermore, H(2)O(2) increased the permeability of a layer of podocytes to albumin: Transmembrane flux for albumin increased five-fold (106.6 ± 5.2 µg/ml vs. 20.2 ± 2.5 µg/ml, P < 0.05, n = 5), and the PKG inhibitor Rp-8-Br-cGMPS (100 µM) prevented the flux increase. These data suggest that oxidative modulation of PKGIα in podocytes plays an important

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